Alcohol abuse is a significant public health burden that is often difficult to manage with current treatment options (NIAAA 1994). Chronic ethanol can induce adaptations in neural circuits that govern emotional behavior and anxiety and may have relevance for ethanol dependence. Serotonin 2c (5HT2c) and corticotrophin-releasing factor (CRF) systems are both dysregulated by chronic ethanol and contribute to the anxiety-inducing effects of ethanol withdrawal (Koob 2001; Overstreet 2003; Knapp 2004; Overstreet 2006; Heilig 2007). The goal of this application is to unravel the molecular basis for 5HT-CRF interactions in the bed nucleus of stria terminalis (BNST), a brain region that was previously shown to be an important neural substrate of anxiety and relapse (Erb 1999). Our central hypothesis is that chronic intermittent ethanol (CIE) increases 5HT2c receptor (5HT2c-R) signaling in the BNST, which in turn activates CRF neurons, causing an increase in anxiety-like behavior that is observed during ethanol withdrawal. Specific Aim 1 is designed to investigate the impact of 5HT2c-R signaling on excitability of CRF neurons in the BNST using ex vivo slice electrophysiology in a recently developed CRF-Ai3 reporter mouse. [Given the potential confounds associated with CRF reporter mice, we include a converging approach that targets the dense CRF projection from the BNST to VTA. Using retrograde tracer beads injected into the VTA of wild-type mice, we will record from putative CRF neurons in the BNST that project to the VTA.] The effects of 5HT2c-R agonists and antagonists on membrane potential and current induced spiking will be examined to determine if 5HT2c-R activation depolarizes CRF neurons in the BNST using both approaches. We will also investigate downstream signaling pathways (e.g. Gq/11/PLC) that may underlie 5HT2c-R effects in CRF neurons. Together, these experiments will clarify the functional interactions between 5HT and CRF systems in the BNST. Specific Aim 2 examines the effects of CIE on 5HT2c-R signaling in the BNST. Using ex vivo slice electrophysiology in CRF-Ai3 reporter [and VTA tracer-injected wild-type mice], we will examine the effects of CIE on excitability and 5HT2c signaling in CRF neurons. Together, these experiments will provide converging results indicating whether CIE leads to functional adaptations in 5HT2c-R systems in the BNST. Specific Aim 3 is designed to clarify the role of 5HT2c-R signaling in the BNST in anxiety-like behavior following CIE. Preliminary data indicates that CIE induces anxiety-like behavior in mice in the social approach test. Given the putative actions of 5HT2c-Rs on CRF neurons in the BNST, we predict that 5HT2c-R antagonists infused into the BNST will alleviate anxiety associated with ethanol withdrawal. The results of this study will indicate whether 5HT2c-R systems in the BNST are a potential target for the treatment of anxiety during ethanol withdrawal. In total, the proposed research will provide essential information concerning 5HT2c- CRF interactions in the BNST and the role that these systems play in alcohol-induced anxiety.